Ocular infectious diseases, including microbial keratitis, conjunctivitis, and endophthalmitis, remain a significant cause of potentially blinding disease. Traditional microbial culture methods are unable to identify causative organisms in many cases; yields for cultures of corneal ulcers, for example, are around 55%. Most infectious organisms causing ocular disease originate in the ocular surface. However, the constituents of this surface have been incompletely characterized to date. Modern molecular biologic methods including recently available deep sequencing methods allow unprecedented analysis of the ocular surface microbiome. Recent pilot studies from both principal investigators laboratories utilizing 16S ribosomal sequencing have demonstrated that 1.) many culture-negative corneal ulcers are associated with novel or unusual organisms, and 2.) the diversity of the normal ocular surface biome is far greater than has been appreciated by traditional, culture-based methods. Taken together, these findings provide an impetus for performing a definitive characterization of the ocular surface (OS) microbiome. Our overarching hypothesis is that, similar to other normally-colonized sites on the body, the OS microbiome is a homeostatically controlled environment which is normally protective of deleterious infection; and that perturbations in this microbiome will predispose to pathologic conditions. In the first Aim of thes studies, two state-of-the-art techniques will be employed in parallel to study the OS microbiome. The first, deep 16S/5.8S sequencing using 454-based pyrosequencing will be used to generate a catalog of bacterial and fungal genera found in the conjunctiva of 100 healthy subjects, diversified for geographic location (from Miami to Seattle), ethnicity, and gender. We will additionally look at the stability of the microbiome across time in a subset of these subjects. The second technique, Biome Representational in Silico Karyotyping, is a novel method for discovery of previously uncharacterized species, utilizing Illumina-based deep sequencing of a defined genomic representation of a metagenomic sample. Using this technique, novel bacteria, viruses, fungi, phage, or parasites can be discovered and molecular diagnostic tests for these organisms can be devised. In Aims 2 and 3, the effect of common clinical scenarios that may disrupt the normal OS microbiome (specifically, treatment with topical antibiotics, topical corticosteroids, and use of soft contact lenses) will be tested using these same techniques. At the conclusions of these studies we anticipate we will have derived a definitive description of the ocular surface microbiome, and determined its variability amongst individuals, its stability within an individual between eyes and over time, and understood the response of this biome to treatment with medications and soft contact lens wear. This information will be essential to subsequent studies aimed at understanding the influence of the microbiome on ocular health. The principal investigators of this study have many years experience in molecular diagnostics. By collaboration, and with inclusion of a bioinformatics expert, expertise in the two complementary approaches to biome characterization can be brought to bear on this problem. Additionally, the geographic separation of the two groups provides excellent opportunity for understanding the effect of locale on the OS biome. PUBLIC HEALTH RELEVANCE: The ocular surface is normally inhabited microbes which do not cause pathologic infection. We hypothesize that this ocular surface microbiome is important to the normal health of the eye. New molecular biologic techniques allow for unprecedented analysis of the components of the normal and disturbed ocular surface microbiome. Using these techniques we propose a definitive analysis of the consistitution, variability, stability, and response to challenge of the ocular surface microbiome in humans.